Intercellular adhesion molecule—2 and its binding ligands

ABSTRACT

The present invention relates to intercellular adhesion molecules (ICAM-2) which are involved in the process through which lymphocytes recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention is directed toward such molecules, screening assays for identifying such molecules and antibodies capable of binding such molecules. The invention also includes uses for adhesion molecules and for the antibodies that are capable of binding them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 08/194,564, filedFeb. 10, 1994, now U.S. Pat. No. 5,565,550; which is a divisional ofapplication Ser. No. 08/089,307, filed Jul. 12, 1993, now abandoned;which is a continuation of U.S. application Ser. No. 07/454,294, filedDec. 22, 1989, now abandoned; which is a continuation-in-part ofapplication Ser. No. 07/321,238, filed Mar. 9, 1989, now abandoned;which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the intercellular adhesion molecule-2(“ICAM-2”) which is involved in the process through which populations oflymphocytes recognize and adhere to cellular substrates so that they maymigrate to sites of inflammation and interact with cells duringinflammatory reactions. The present invention additionally relates toligand molecules capable of binding to ICAM-2 intercellular adhesionmolecules, and to uses for the intercellular adhesion molecule, and theligand molecules.

2. Description of the Related Art

Leukocytes must be able to attach to cellular substrates in order toproperly defend the host against foreign invaders such as bacteria orviruses. An excellent review of the defense system is provided by Eisen,H. W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, Pa.(1980), pp. 290-295 and 381-418). They must be able to attach toendothelial cells so that they can migrate from circulation to sites ofongoing inflammation. Furthermore, they must attach toantigen-presenting cells so that a normal specific immune response canoccur, and finally, they must attach to appropriate target cells so thatlysis of virally-infected or tumor cells can occur.

Recently, leukocyte surface molecules involved in mediating suchattachments were identified using hybridoma technology. Briefly,monoclonal antibodies directed against human T-cells (Davignon, D. etal., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleencells (Springer, T. et al. Eur. J. Immunol. 2:301-306 (1979)) wereidentified which bound to leukocyte surfaces and inhibited theattachment related functions described above (Springer, T. et al., Fed.Proc. 44:2660-2663 (1985)). The molecules identified by those antibodieswere called Mac-1 and Lymphocyte Function-associated Antigen-1 (LFA-1).Mac-1 is a heterodimer found on macrophages, granulocytes and largegranular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes(Springer, T. A. et al. Immunol. Rev. 68:111-135 (1982)). These twomolecules, plus a third molecule, p150,95 (which has a tissuedistribution similar to Mac-1) play a role in cellular adhesion (Keizer,G. et al., Eur. J. Immunol. 15:1142-1147 (1985)).

The above-described leukocyte molecules were found to be members of arelated family of glycoproteins (Sanchez-Madrid, F. et al., J. Exper.Med. 158:1785-1803 (1983); Keizer, G. D. et al., Eur. J. Immunol.15:1142-1147 (1985)), termed the “CD-18 family” of glycoproteins. Thisglycoprotein family is composed of heterodimers having one alpha chainand one beta chain. Although the alpha chain of each of the antigensdiffered from one another, the beta chain was found to be highlyconserved (Sanchez-Madrid, F. et al., J. Exper. Med. 158:1785-1803(1983)). The beta chain of the glycoprotein family (sometimes referredto as “CD18”) was found to have a molecular weight of 95 kd whereas thealpha chains were found to vary from 150 kd to 180 kd (Springer, T.,Fed. Proc. 44:2660-2663 (19853). Although the alpha subunits of themembrane proteins do not share the extensive homology shared by the betasubunits, close analysis of the alpha subunits of the glycoproteins hasrevealed that there are substantial similarities between them. Reviewsof the similarities between the alpha and beta subunits of the LFA-1related glycoproteins are provided by Sanchez-Madrid, F. et al., (J.Exper. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983)).

A group of individuals has been identified who are unable to expressnormal amounts of any member of this adhesion protein family on theirleukocyte cell surface (Anderson, D. C. et al., Fed. Proc. 44:2671-2677(1985); Anderson, D. C. et al., J. Infect. Dis. 152:668-689 (1985)).Lymphocytes from these patients displayed in vitro defects similar tonormal counterparts whose CD-18 family of molecules had been antagonizedby antibodies. Furthermore, these individuals were unable to mount anormal immune response due to an inability of their cells to adhere tocellular substrates (Anderson, D. C. et al., Fed. Proc. 44:2671-2677(1985); Anderson, D. C. et al., J. Infect. Dis. 152:668-689 (1985)).These data show that immune reactions are mitigated when lymphocytes areunable to adhere in a normal fashion due to the lack of functionaladhesion molecules of the CD-18 family.

Thus, in summary, the ability of leukocytes to maintain the health andviability of an animal requires that they be capable of adhering toother cells (such as endothelial cells). This adherence has been foundto require cell-cell contacts which involve specific receptor moleculespresent on the cell surface of the leukocytes. These receptors enable aleukocyte to adhere to other leukocytes or to endothelial, and othernon-vascular cells. The cell surface receptor molecules have been foundto be highly related to one another. Humans whose leukocytes lack thesecell surface receptor molecules exhibit chronic and recurringinfections, as well as other clinical symptoms including defectiveantibody responses.

Since leukocyte adhesion is involved in the process through whichforeign tissue is identified and rejected, an understanding of thisprocess is of significant value in the fields of organ transplantation,tissue grafting, allergy and oncology.

SUMMARY OF THE INVENTION

The present invention relates to Intercellular Adhesion Molecule-2(ICAM-2) as well as to its functional derivatives. The inventionadditionally pertains to antibodies and fragments of antibodies capableof inhibiting the function of ICAM-2, and to other inhibitors of ICAM-2function. The invention additionally includes diagnostic and therapeuticuses for all of the above-described molecules.

In detail, the invention includes the intercellular adhesion moleculeICAM-2, or a functional derivative thereof, substantially free ofnatural contaminants.

The invention further pertains to ICAM-2 which contains at least onepolypeptide selected from the group consisting of:

(a) -S-S-F-G-Y-R-T-L-T-V-A-L-;

(b) -D-E-K-V-F-E-V-H-V-R-P-K-;

(c) -G-S-L-E-V-N-C-S-T-T-C-N-;

(d) -H-Y-L-V-S-N-I-S-H-T-D-V-;

(e) -S-M-N-S-N-V-S-V-Y-Q-P-P-;

(f) -F-T-I-E-C-R-V-P-T-V-E-P-;

(g) -G-N-E-T-L-H-Y-E-T-F-G-K-;

(h) -T-A-T-F-N-S-T-A-D-R-E-D-;

(i) -H-R-N-F-S-C-L-A-V-L-D-L-;

(j) -M-V-I-I-V-T-V-V-S-V-L-L-;

(k) -S-L-F-V-T-S-V-L-L-C-F-I-; and

(l) -M-G-T-Y-G-V-R-A-A-W-R-R-.

The invention also provides a recombinant or synthetic. DNA moleculecapable of encoding, or of expressing, ICAM-2 or a functional derivativethereof.

The invention additionally provides an antibody, and especially amonoclonal antibody, capable of binding to a molecule selected from thegroup consisting of ICAM-2, and a functional derivative of ICAM-2.

The invention also provides a hybridoma cell capable of producing theabove-described monoclonal antibody.

The invention includes a method for producing a desired hybridoma cellthat produces an antibody which is capable of binding to ICAM-2, or itsfunctional derivative, which comprises the steps:

(a) immunizing an animal with an imunogen selected from the groupconsisting of: a cell expressing ICAM-2, a membrane of a cell expressingICAM-2, ICAM-2, ICAM-2 bound to a carrier, a peptide fragment of ICAM-2,and a peptide fragment of ICAM-2 bound to a carrier,

(b) fusing the spleen cells of the animal with a myeloma cell line,

(c) permitting the fused spleen and myeloma cells to form antibodysecreting hybridoma cells, and

(d) screening the hybridoma cells for the desired hybridoma cell that iscapable of producing an antibody capable of binding to ICAM-2.

The invention also provides a method for treating inflammation resultingfrom a response of the specific defense system in a mammalian subjectwhich comprises providing to a subject in need of such treatment anamount of an anti-inflammatory agent sufficient to suppress theinflammation; wherein the anti-inflammatory agent is selected from thegroup consisting of: an antibody capable of binding to ICAM-2; afragment of an antibody, the fragment being capable of binding toICAM-2; ICAM-2; a functional derivative of ICAM-2; and anon-immunoglobulin antagonist of ICAM-2 other than ICAM-1, or a memberof the CD-18 family of molecules.

The invention also includes a method of suppressing the metastasis of ahematopoietic tumor cell, the cell having a member of the CD-18(especially LFA-1) for migration, which method comprises providing to apatient in need of such treatment an amount of an agent sufficient tosuppress the metastasis; wherein the agent is selected from the groupconsisting of: an antibody capable of binding to ICAM-2; atoxin-derivatized antibody capable of binding to ICAM-2; a fragment ofan antibody, the fragment being capable of binding to ICAM-2; atoxin-derivatized fragment of an antibody, the fragment being capable ofbinding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; atoxin-derivatized ICAM-2; and a toxin-derivatized functional derivativeof ICAM-2; and a non-immunoglobulin antagonist of ICAM-2 other thanICAM-1, or a member of the CD-18 family of molecules.

The invention also includes a method of suppressing the growth of anICAM-2-expressing tumor cell which comprises providing to a patient inneed of such treatment an amount of an agent sufficient to suppress thegrowth, wherein the agent is selected from the group consisting of: anantibody capable of binding to ICAM-2; a toxin-derivatized antibodycapable of binding to ICAM-2; a fragment of an antibody, the fragmentbeing capable of binding to ICAM-2; a toxin-derivatized fragment of anantibody, the fragment being capable of binding to ICAM-2; ICAM-2; afunctional derivative of ICAM-2; a non-immunoglobulin antagonist ofICAM-2 other than ICAM-1, or a member of the CD-18 family of molecules;a toxin-derivatized member of the CD-18 family of molecules; and atoxin-derivatized functional derivative of a member of the CD-18 familyof molecules.

The invention also provides a method for detecting the presence of acell expressing ICAM-2 which comprises:

(a) incubating the cell or an extract of the cell in the presence of anucleic acid molecule, the nucleic acid molecule being capable ofhybridizing to ICAM-2 mRNA; and

(b) determining whether the nucleic acid molecule has become hybridizedto a complementary nucleic acid molecule present in said cell or in saidextract of said cell.

The invention also provides a phamaceutical composition comprising:

(a) an anti-inflammatory agent selected from the group consisting of: anantibody capable of binding to ICAM-2; a fragment of an antibody body,the fragment being capable of binding to ICAM-2; ICAM-2; a functionalderivative of ICAM-2; and a non-immunoglobulin antagonist of ICAM-2other than ICAM-1, or a member of the CD-18 family of molecules, eitheralone, or in combination with (b) an immunosuppressive agent.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and 1 b show binding of transfected COS cells expressing ICAM-1and ICAM-2 to LFA-1-coated plastic. FIG. 1a) COS cells transfected withICAM-1 cDNA were panned on LFA-1-coated plates and the expression ofICAM-1 was analyzed by indirect immunofluorescence flow cytometry withanti-ICAM-1 monoclonal antibody RR1/1 as primary MAb. Unpanned cells(dotted line), non-adherent cells (dashed line), adherent cells (solidline). FIG. 1b) ⁵¹Cr labelled transfected COS cells expressing ICAM-1 orICAM-2 were bound to LFA-1-coated plastic in the presence of MAb.

FIGS. 2-1, 2-2, 2-3 and 2-4 show the nucleotide and amino acid sequenceof ICAM-2. The amino acid sequence is numbered beginning with the firstresidue following the predicted cleavage site of the signal peptide. Thehydrophobic putative signal peptide and transmembrane sequences (TM) areunderlined. Potential N-linked glycosylation sites are boxed. Theputative polyadenylation signal AATACA is overlined. Potential N-linkedglycosylation sites are boxed. The putative polyadenylation signalAATACA is overlined. Both strands of the ICAM-2 cDNA were sequencedwithin CDM8 by sequential synthesis of complementary oligonucleotideprimers and dideoxynucleotide chain termination sequencing (Sanger, F.et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) according to themanufacturer's recommendations (Sequenase, U.S. Biochemical).

FIGS. 3a-3 c show the results of RNA and DNA hybridization analyses.Northern (FIGS. 3a and 3 b) and Southern (FIG. 3c) blots were hybridizedto the 1.1 kb ³²P labeled ICAM-2 cDNA (FIGS. 3a and 3 c) andrehybridized to the 3 kb ³²P labeled ICAM-1 cDNA (FIG. 3b). (FIGS. 3aand 3 b) 6 μg of poly(A)⁺ RNA from the Burkitt lymphoma cell line, Ramos(lane 1), endothelial cells (lane 2), endothelial cells stimulated forthree hours with LPS (lane 3), an EBV immortalized B-lymphoblastoid cellline, BBN (lane 4), epithelial carcinoma cell line, HeLa (lane 5), Tlymphoma cell lines, Jurkat (lane 6) and SKW-3 (lane 7), and apromonocyte cell line, U937 (lane 8). (FIG. 3c) 6 μg of genomic DNA fromB cell lines BL-2 (lanes 1 and 4), ER-LCL (lanes 2 and 5) and Raji(lanes 3 and 6) digested with EcoRI (lanes 1-3) or HindIII (lanes 4-6).ICAM-2 and ICAM-1 mRNAs are indicated by arrows.

FIGS. 4-1 and 4-2 show ICAM-2 homology to ICAM-1. The entire 201 residueextracellular sequence of ICAM-2 was aligned with ICAM-1 residues 1-185using the ALIGN program (Dayhoff, M.O. et al., Methods Enzymol.91:524-545 (1983)) and by inspection. ICAM-2 residues are numbered.Identities are boxed. D1 and D2 indicate the boundary of Ig-like domainsof ICAM-2 and ICAM-1. β strand predictions (Chou, P. Y. et al.,Biochemistry 13:211-245 (1974)) of ICAM-2 are overlined and those ofICAM-1 are underlined.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention relates to the discovery of anatural binding ligand to LFA-1. Molecules such as those of CD-18family, which are involved in the process of cellular adhesion arereferred to as “adhesion molecules.”

I. LFA-1 and ICAM-1

The leukocyte adhesion molecule LFA-1 mediates a wide range oflymphocyte, monocyte, natural killer cell, and granulocyte interactionswith other cells in immunity and inflammation (Springer, T. A. et al.,Ann. Rev. Immunol. 5:223-252 (1987)).

LFA-1 is a receptor for intercellular adhesion molecule 1 (ICAM-1), asurface molecule is constitutively expressed on some tissues and inducedon others in inflammation (Marlin, S. D. et al., Cell 51:813-819 (1987);Dustin, M. L. et al., J. Immunol. 137:245-254 (1986); Dustin, M. L. etal., Immunol. Today 9:213-215 (1988); U.S. patent application Ser. No.07/019,440, filed Feb. 26, 1987 and U.S. patent application Ser. No.07/250,446, filed Sep. 28, 1988, both applications herein incorporatedby reference).

LFA-1 functions in both antigen-specific-and antigen-independent Tcytotoxic, T helper, natural killer, granulocyte and monocyteinteractions with other cell types (Springer, T. A. et al., Ann. Rev.Immunol. 5:223-252 (1987); Kishimoto, T. K. et al., Adv. Immunol. (1988,in press)). LFA-1 is a leukocyte integrin, with noncovalently associatedα and β glycoprotein subunits of 180 and 95 kD.

ICAM-1 is a single chain glycoprotein varying in mass on different celltypes from 76-114 kD, and is a member of the Ig superfamily with fiveC-like domains (Dustin, M. L. et al., Immunol. Today 9:213-215 (1988);Staunton, D. E. et al., Cell 52:925-933 (1988); Simmons, D. et al.,Nature 331:624-627 (1988)). ICAM-1 is highly inducible with cytokinesincluding IFN-γ, TNF, and IL-1 on a wide range of cell types (Dustin, M.L. et al., Immunol. Today 9:213-215 (1988)). Induction of ICAM-1 onepithelial cells, endothelial cells, and fibroblasts mediates LFA-1dependent adhesion of lymphocytes (Dustin, M. L. et al., J. Immunol.137:245-254 (1986); Dustin, M. L. et al., J. Cell. Biol. 107:321-331(1988); Dustin, M. L. et al., J. Exp. Med. 167:1323-1340 (1988)).Adhesion is blocked by pretreatment of lymphocytes with LFA-1 MAb orpretreatment of the other cell with ICAM-1 MAb (Dustin, M. L. et al., J.Immunol. 137:245-254 (1986); Dustin, M. L. et al., J. Cell. Biol.107:321-331 (1988); Dustin, M. L. et al., J. Exp. Med. 167:1323-1340(1988)). Identical results with purified ICAN-1 in artificial membranesor on Petri dishes demonstrate that LFA-1 and ICAM-1 are receptors forone another (Marlin, S. D. et al., Cell 51:813-819 (1987); Makgoba, M.W. et al., Nature 331:86-88 (1988)). For clarity, they are referred toherein as “receptor” and “ligand,” respectively. Further descriptions ofICAM-1 are provided in U.S. patent applications Ser. Nos. 07/045,963;07/115,798; 07/155,943; 07/189,815 or 07/250,446, all of whichapplications are herein incorporated by reference in their entirety.

II. ICAM-2

A second LFA-1 ligand, distinct from ICAM-1, has been postulated(Rothlein, R. et al., J. Immunol. 137:1270-1274 (1986); Makgoba, M. W.et al., Eur. J. Immunol. 18:637-640 (1988); Dustin, M. L. et al., J.Cell. Biol. 107:321-331 (1988)). The present invention concerns thissecond ligand, designated “ICAM-2” (for “Intercellular AdhesionMolecule—2”).

ICAM-2 differs from ICAM-1 in cell distribution and in a lack ofcytokine induction. ICAM-2 is an integral membrane protein with 2Ig-like domains, whereas ICAM-1 has 5 Ig-like domains (Staunton, D. E.et al., Cell 52:925-933 (1988); Simmons, D. et al., Nature 331:624-627(1988)). Remarkably, ICAM-2 is much more closely related to the two mostN-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2is to other members of the Ig superfamily, demonstrating a subfamily ofIg-like ligands which bind the same integrin family receptor.

III. cDNA Cloning of ICAM-2

Any of a variety of procedures may be used to clone the ICAM-2 gene. Onesuch method entails analyzing a shuttle vector library of cDNA inserts(derived from an ICAM-2 expressing cell) for the presence of an insertwhich contains the ICAM-2 gene. Such an analysis may be conducted bytransfecting cells with the vector and then assaying for ICAM-2expression.

ICAM-2 cDNA is preferably identified when a novel modification of theprocedure of Aruffo and Seed (Seed, B. et al., Proc. Natl. Acad. Sci.USA 84:3365-3369 (1987)) is employed for identifying ligands of adhesionmolecules. In this method, a cDNA library is prepared from cells whichexpress ICAM-2 (such as endothelial cells or Ramos, BBN Blymphoblastoid, U937 monocytic, or SKW3 lymphoblastoid, cell lines).Preferrably, the CDNA library is prepared from endothelial cells. Thislibrary is used to transfect cells which do not normally express ICAM-2(such as COS cells). The transfected cells are introduced into a petridish which has been previously coated with LFA-1. COS cells which havebeen transfected with either ICAM-1 or ICAM-2 encoding sequences, andwhich express either of these ligands on their cell surfaces will adhereto the LFA-1 on the surface of the petri dish. Non-adherent cells arewashed away, and the adherent cells are then removed from the petri dishand cultured. The recombinant ICAM-1 or ICAM-2 expressing sequences inthese cells is then removed, and sequenced to determine whether itencodes ICAM-1 or ICAM-2.

In a preferred embodiment of the above-described method, anti-ICAM-1antibody is added to the petri dish in order to prevent the adherence ofICAM-1 expressing cells. Binding of ICAM-2 transfected COS cells toLFA-1 is inhibited by EDTA and anti-LFA-1 monoclonal antibody (“MAb”),but is not inhibited by anti-ICAM-1 MAb. Thus, in this embodiment, theICAM-1 expressing cells are unable to adhere to the petri dish throughICAM-1 and are therefore mostly washed away with all of the othernonadherent cells. As a result, only cells expressing ICAM-2 are able toadhere to the petri dish.

Thus, cDNA clones are screened by expression in COS cells, and bypanning for ligand-bearing COS cells using functionally-active, purifiedLFA-1 which has been previously bound to plastic Petri dishes. Afterpanning, nonadherent cells are depleted of ICAM-2⁺ cells, whereasadherent cells, released from LFA-1-coated plastic by EDTA, are almostcompletely ICAM-2⁺. Adherence of ICAM-1⁺ cells to LFA-1-coated plasticmay be inhibited with RR1/1 anti-ICAM-1 MAb.

Thus, in accordance with this method for cloning CDNA for ICAM-2, a cDNAlibrary is prepared from endothelial cells, which demonstrate both theICAM-1-dependent and ICAM-1-independent components of LFA-1-dependentadhesion (Dustin, M. L. et al., J. Cell. Biol. 107:-321-331 (1988))using a suitable plasmid, such as the plasmid vector CDM8. TransfectedCOS cells are incubated in LFA-1-coated petri dishes with anti-ICAM-1MAb present to reduce the probability of isolating ICAM-1 cDNA's.Adherent cells are eluted with EDTA and plasmids are isolated andamplified in E. coli. After approximately three cycles of transfection,adherence, and plasmid isolation and one size fractionation, plasmidsmay be analyzed by restriction endonuclease digestion. Approximately ⅓of plasmids having inserts greater than 1.0 kb, when introduced into COScells by transfection, yielded adherence to LFA-1.

Alternatively, a cDNA clone of ICAM-2 can be obtained by using thegenetic code (Watson, J. D., In: Molecular Biology of the Gene, 3rd Ed.,W. A. Benjamin, Inc., Menlo Park, Calif. (1977)-, pp. 356-357) todetermine the sequence of a polynucleotide capable of encoding theICAM-2 protein.

A clone of the ICAM-2 cDNA can also be obtained by identifying the aminoacid sequences of peptide fragments of the ICAM-2 protein, and thenusing the genetic code to construct oligonucleotide probe moleculescapable of encoding the ICAM-2 peptide. The probes are then used todetect (via hybridization) those members of a cDNA library (preparedfrom cDNA of ICAM-2 expressing cells) which encode the ICAM-2 protein.

Techniques such as, or similar to, those described above havesuccessfully enabled the cloning of genes for human aldehydedehydrogenases (Hsu, L. C. et al., Proc. Natl. Acad. Sci. USA82:3771-3775 (1985)), fibronectin (Suzuki, S. et al., Eur. Mol. Biol.Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter,P. et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-typeplasminogen activator (Pennica, D. et al., Nature 301:214-221 (1983))and human term placental alkaline phosphatase complementary DNA (Kam, W.et al., Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

In yet another alternative way of cloning the ICAM-2 gene, a library ofexpression vectors is prepared by cloning DNA or, more preferably cDNA,from a cell capable of expressing ICAM-2 into an expression vector. Thelibrary is then screened for members capable of expressing a proteinwhich binds to anti-ICAM-2 antibody, and which has a nucleotide sequencethat is capable of encoding polypeptides that have the same amino acidsequence as ICAM-2 or fragments of ICAM-2.

The cloned ICAM-2 gene, obtained through the use of any of the methodsdescribed above, may be operably linked to an expression vector, andintroduced into bacterial, or eukaryotic cells to produce ICAM-2protein. Techniques for such manipulations are disclosed by Maniatis, T.et al., supra, and are well known in the art.

IV. The Agents of the Present Invention: ICAN-2 and Its FunctionalDerivatives, Agonists and Antagonists

The present invention is directed toward ICAM-2, its “functionalderivatives,” and its “agonists” and “antagonists.”

A. Functional Derivatives of ICAN-2

A “functional derivative” of ICAM-2 is a compound which possesses abiological activity (either functional or structural) that issubstantially similar to a biological activity of ICAM-2. The term“functional derivatives” is intended to include the “fragments,”“variants,” “analogs,” or “chemical derivatives” of a molecule.

A “fragment” of a molecule such as ICAM-2, is meant to refer to anypolypeptide subset of the molecule. Fragments of ICAM-2 which haveICAM-2 activity and which are soluble (i.e not membrane bound) areespecially preferred.

A “variant” of a molecule such as ICAM-2 is meant to refer to a moleculesubstantially similar in structure and function to either the entiremolecule, or to a fragment thereof.

An “analog” of a molecule such as ICAM-2 is meant to refer to a moleculesubstantially similar in function to either the entire molecule or to afragment thereof.

A molecule is said to be “substantially similar” to another molecule ifboth molecules have substantially similar structures or if bothmolecules possess a similar biological activity. Thus, provided that twomolecules possess a similar activity, they are considered variants asthat term is used herein even if the structure of one of the moleculesis not found in the other, or if the sequence of amino acid residues isnot identical.

As used herein, a molecule is said to be a “chemical derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half life, etc. Themoieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences (1980).

“Toxin-derivatized” molecules constitute a special class of “chemicalderivatives.” A “toxin-derivatized” molecule is a molecule (such asICAM-2 or an antibody) which contains a toxin moiety. The binding ofsuch a molecule to a cell brings the toxin moiety into close proximitywith the cell and thereby promotes cell death. Any suitable toxin moietymay be employed; however, it is preferable to employ toxins such as, forexample, the ricin toxin, the cholera toxin, the diphtheria toxin,radioisotopic toxins, membrane-channel-forming toxins, etc. Proceduresfor coupling such moieties to a molecule are well known in the art.

Functional derivatives of ICAM-2 having up to about 100 residues may beconveniently prepared by in vitro synthesis. If desired, such fragmentsmay be modified by reacting targeted amino acid residues of the purifiedor crude protein with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. The resultingcovalent derivatives may be used to identify residues important forbiological activity.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneire used to form 0-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking anICAM-2 functional derivative molecule to a water-insoluble supportmatrix or surface for use in the method for cleaving an ICAM-2functional derivatives fusion polypeptide to release and recover thecleaved polypeptide. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Functional derivatives of ICAM-2 having altered amino acid sequences canalso be prepared by mutations in the DNA. The nucleotide sequence whichencodes the ICAM-2 gene is shown in FIGS. 2-1, 2-2, 2-3 and 2-4. Suchvariants include, for example, deletions from, or insertions orsubstitutions of, residues within the amino acid sequence shown in FIGS.2-1, 2-2, 2-3 and 2-4. Any combination of deletion, insertion, andsubstitution may also be made to arrive at the final construct, providedthat the final construct possesses the desired activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure (seeEP Patent Application Publication No. 75,444).

At the genetic level, these functional derivatives ordinarily areprepared by site-directed mutagenesis of nucleotides in the DNA encodingthe ICAM-2 molecule, thereby producing DNA encoding the functionalderivative, and thereafter expressing the DNA in recombinant cellculture. The functional derivatives typically exhibit the samequalitative biological activity as the naturally occurring analog. Theymay, however, differ substantially in such characteristics with respectto the normally produced ICAM-2 molecule.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed ICAM-2 functional derivatives screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example, site-specific mutagenesis.

Preparation of an ICAM-2 functional derivative molecule in accordanceherewith is preferably achieved by site-specific mutagenesis of DNA thatencodes an earlier prepared functional derivatives or a nonvariantversion of the protein. Site-specific mutagenesis allows the productionof ICAM-2 functional derivatives through the use of specificoligonucleotide sequences that encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 20 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues on both sides of the junctionof the sequence being altered. In general, the technique ofsite-specific mutagenesis is well known in the art, as exemplified bypublications such as Adelman et al., DNA 2:183 (1983), the disclosure ofwhich is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phagesare readily commercially available and their use is generally well knownto those skilled in the art. Alternatively, plasmid vectors that containa single-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153:3 (1987)) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells, such as JM101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues, and typically arecontiguous. Deletions may also comprise an immunoglobulin domain, suchas domains 1 or 2 of ICAM-2. Amino acid sequence insertions includeamino and/or carboxyl-terminal fusions of from one residue topolypeptides of essentially unrestricted length, as well asintrasequence insertions of single or multiple amino acid residues.Intrasequence insertions (i.e., insertions within the complete ICAM-2molecule sequence) may range generally from about 1 to 10 residues, morepreferably 1 to 5. An example of a terminal insertion includes a fusionof a signal sequence, whether heterologous or homologous to the hostcell, to the N-terminus of the molecule to facilitate the secretion ofthe ICAM-2 functional derivative from recombinant hosts.

The third group of functional derivatives are those in which at leastone amino acid residue in the ICAM-2 molecule, and preferably, only one,has been removed and a different residue inserted in its place. Suchsubstitutions preferably are made in accordance with the following Tablewhen it is desired to modulate finely the characteristics of the ICAM-2molecule.

TABLE 1 Original Residue Exemplary Substitutions Ala gly; ser Arg lysAsn gln; his Asp glu Cys ser Gln asn Glu asp Gly ala; pro His asn; glnIle leu; val Leu ile; val Lys arg; gln; glu Met leu; tyr; ile Phe met;leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in functional or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected are those in which (a) glycine and/or proline issubstituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; or (e) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having such a side chain, e.g., glycine.

Most deletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of the ICAM-2molecule. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. For example, a functional derivative typicallyis made by site-specific mutagenesis of the native ICAM-2molecule-encoding nucleic acid, expression of the variant nucleic acidin recombinant cell culture, and, optionally, purification from the cellculture, for example, by immunoaffinity adsorption on an anti-ICAM-2molecule antibody column (to absorb the functional derivative by bindingit to at least one remaining immune epitope).

Mutations designed to increase the affinity of ICAM-2 may be guided bythe introduction of the amino acid residues which are present athomologous positions in ICAM-1. Similarly, such mutant ICAM-2 moleculesmay be prepared which lack N-linked CHO at homologous positions inICAM-1.

The activity of the cell lysate or purified ICAM-1 molecule functionalderivative is then screened in a suitable screening assay for thedesired characteristic. For example, a change in the immunologicalcharacter of the functional derivative, such as affinity for a givenantibody, is measured by a competitive type immunoassay. Changes inimmunomodulation activity are measured by the appropriate assay.Modifications of such protein properties as redox or thermal stability,biological half-life, hydrophobicity, susceptibility to proteolyticdegradation or the tendency to aggregate with carriers or into multimersare assayed by methods well known to the ordinarily skilled artisan.

B. Agonists and Antagonists of ICAM-2

An “agonist” of ICAM-2 is a compound which enhances or increases theability of ICAM-2 to carry out any of its biological functions. Anexample of such an agonist is an agent which increases the ability ofICAM-2 to bind to a cellular receptor or viral protein.

An “antagonist” of ICAM-2 is a compound which diminishes or prevents theability of ICAM-2 to carry out any of its biological functions. Examplesof such antagonists include ICAM-1, functional derivatives of ICAM-1,anti-ICAM-2 antibody, anti-LFA-1 antibody, etc.

The cellular aggregation assays described in U.S. patent applicationsSer. Nos. 07/045,963; 07/115,798; 07/155,943; 07/189,815 or 07/250,446,all of which applications have been herein incorporated by reference intheir entirety, are capable of measuring LFA-1 dependent aggregation,and may be employed to identify agents which affect the the extent ofICAM-2/LFA-1 aggregation. Thus, such assays may be employed to identifyagonists and antagonists of ICAM-2. Antagonists may act by impairing theability of LFA-1 or of ICAM-2 to mediate aggregation. Additionally,non-immunoglobulin (i.e., chemical) agents may be examined, using theabove-described assay, to determine whether they are agonists orantagonists of ICAM-2/LFA-1 aggregation.

C. Anti-ICAM-2 Antibody

The preferred immunoglobulin antagonist of the present invention is anantibody to ICAM-2. Suitable antibodies can be obtained in any of avariety of ways.

An antigenic molecule such as ICAM-2 is naturally expressed on thesurfaces of lymphocytes. Thus, the introduction of such cells into anappropriate animal, as by intraperitoneal injection, etc., will resultin the production of antibodies capable of binding to ICAM-2 or membersof the CD-18 family of molecules. If desired, the serum of such ananimal may be removed and used as a source of polyclonal antibodiescapable of binding these molecules.

Alternatively, anti-ICAM-2 antibodies may be produced by adaptation ofthe method of Selden, R. F. (European Patent Application Publication No.289,034) or Selden R. F. et al. (Science 236:714-718 (1987)). Inaccordance with such an adaptation of this method, the cells of asuitable animal (i.e. such as, for example, a mouse, etc.) aretransfected with a vector capable of expressing either the intact ICAM-2molecule, or a fragment of ICAM-2. The production of ICAM-2 in thetransfected cells of the animal will elicit an immune response in theanimal, and lead to the production of anti-ICAM-2 antibodies by theanimal.

Alternatively, anti-ICAM-2 antibodies may be made by introducing ICAM-2,or peptide fragments thereof, into an appropriate animal. The immunizedanimal will produce polyclonal antibody in response to such exposure.The use of peptide fragments of ICAM-2 permits one to obtain regionspecific antibodies which are reactive only with the epitope(s)contained in the peptide fragments used to immunize the animals.

It is, however, preferable to remove splenocytes from animals (immunizedin either of the ways described above), to fuse such spleen cells with amyeloma cell line and to permit such fusion cells to form a hybridomacell which secretes monoclonal antibodies capable of binding ICAM-2.

The hybridoma cells, obtained in the manner described above may bescreened by a variety of methods to identify desired hybridoma cellsthat secrete antibody capable of binding to ICAM-2. In a preferredscreening assay, such molecules are identified by their ability toinhibit the aggregation of ICAM-2-expressing, ICAM-1-non-expressingcells. Antibodies capable of inhibiting such aggregation are thenfurther screened to determine whether they inhibit such aggregation bybinding to ICAM-2, or to a member of the CD-18 family of molecules. Anymeans capable of distinguishing ICAM-2 from the CD-18 family ofmolecules may be employed in such a screen. Thus, for example, theantigen bound by the antibody may be analyzed as by immunoprecipitationand polyacrylamide gel electrophoresis. It is possible to distinguishbetween those antibodies which bind to members of the CD-18 family ofmolecules from those which bind ICAM-2 by screening for the ability ofthe antibody to bind to cells which express LFA-1, but not ICAM-2 (orvice-versa). The ability of an antibody to bind to a cell expressingLFA-1 but not ICAM-2 may be detected by means commonly employed by thoseof ordinary skill. Such means include immunoassays (especially thoseusing immunoflorescence), cellular agglutination, filter bindingstudies, antibody precipitation, etc.

In addition to the above-described functional derivatives of ICAM-2,other agents which may be used in accordance of the present invention inthe treatment of viral infection or inflammation include antibody toICAM-2, anti-idiotypic antibodies to anti-ICAM-2 antibodies, andreceptor molecules, or fragments of such molecules, which are capable ofbinding to ICAM-2.

The antibodies to ICAM-2 (or functional derivatives of ICAM-2) which maybe employed may be either polyclonal or monoclonal.

The anti-idiotypic antibodies of interest to the present invention arecapable of binding in competion with (or to the exclusion of) ICAM-2.Such antibodies can be obtained, for example, by raising antibody to ananti-ICAM-2 antibody, and then screening the antibody for the ability tobind a natural binding ligand of ICAM-2.

Since molecules of the CD-18 family are able to bind to ICAM-2,administration of such molecules (for example as heterodimers havingboth alpha and beta subunits, or as molecules composed of only an alpha,or a beta subunit, or as molecules having fragments of either or bothsubunits) is able to compete with (or exclude) HRV for binding toICAM-21 present on cells.

The anti-aggregation antibodies of the present invention may beidentified and titered in any of a variety of ways. For example, one canmeasure the ability of the antibodies to differentially bind to cellswhich express ICAM-2 (such as activated endothelial cells), and theirinability to bind to cells which fail to express ICAM-2. Suitable assaysof cellular aggregation are those described in U.S. patent applicationsSer. Nos. 07/045,963; 07/115,798; 07/155,943; 07/189,815 or 07/250,446,all of which applications have been herein incorporated by reference intheir entirety. Alternatively, the capacity of the antibodies to bind toICAM-2 or to peptide fragments of ICAM-2 can be measured. As will bereadily appreciated by those of ordinary skill, the above assays may bemodified, or performed in a different sequential order to provide avariety of potential screening assays, each of which is capable ofidentifying and discriminating between antibodies capable of binding toICAM-1 versus members of the CD-18 family of molecules.

In a more preferred method, antibody can be selected for its ability tobind to COS cells expressing ICAM-2, but not to COS cells which do notexpress ICAM-2.

D. Preparation of the Agents of the Present Invention

The agents of the present invention may be obtained by natural processes(such as, for example, by inducing an animal, plant, fungi, bacteria,etc., to produce a non-immunoglobulin antagonist of ICAM-2, or byinducing an animal to produce polyclonal antibodies capable of bindingto ICAM-2); by synthetic methods (such as, for example, by using theMerrifield method for synthesizing polypeptides to synthesize ICAM-2,functional derivatives of ICAM-2, or protein antagonists of ICAM-2(either immunoglobulin or non-immunoglobulin)); by hybridoma technology(such as, for example, to produce monoclonal antibodies capable ofbinding to ICAM-2); or by recombinant technology (such as, for example,to produce the agents of the present invention in diverse hosts (i.e.,yeast, bacteria, fungi, cultured mammalian cells, etc.), or fromrecombinant plasmids or viral vectors). The choice of which method toemploy will depend upon factors such as convenience, desired yield, etc.It is not necessary to employ only one of the above-described methods,processes, or technologies to produce a particular anti-inflammatoryagent; the above-described processes, methods, and technologies may becombined in order to obtain a particular agent.

V. Uses of ICAM-2, and Its Functional Derivatives, Agonists andAntagonists

A. Suppression of Inflammation

One aspect of the present invention derives from the ability of ICAM-2and its functional derivatives to interact with receptors of the CD-18family of molecules, especially LFA-1 or with viral proteins (such asthe proteins of the rhinovirus, etc.). By virtue of the ability ofICAM-2 to interact with members of the CD-18 family of glycoproteins, itmay be used to suppress (i.e. to prevent, or attenuate) inflammation.

The term “inflammation,” as used herein, is meant to include both thereactions of the specific defense system, and the reactions of thenon-specific defense system.

As used herein, the term “specific defense system” is intended to referto that component of the immune system that reacts to the presence ofspecific antigens. Inflammation is said to result from a response of thespecific defense system if the inflammation is caused by, mediated by,or associated with a reaction of the specific defense system. Examplesof inflammation resulting from a response of the specific defense systeminclude the response to antigens such as rubella virus, autoimmunediseases, delayed type hypersensitivity response mediated by T-cells (asseen, for example in individuals who test “positive” in the Mantauxtest), etc. Chronic Inflammatory diseases and the rejection oftransplanted tissue and organs are further examples of inflammatoryreactions of the specific defense system.

As used herein, a reaction of the “non-specific defense system” isintended to refer to a reaction mediated by leukocytes incapable ofimmunological memory. Such cells include granulocytes and macrophages.As used herein, inflammation is said to result from a response of thenon-specific defense system, if the inflammation is caused by, mediatedby, or associated with a reaction of the non-specific defense system.Examples of inflammation which result, at least in part, from a reactionof the non-specific defense system include inflammation associated withconditions such as: adult respiratory distress syndrome (ARDS) ormultiple organ injury syndromes secondary to septicemia or trauma;reperfusion injury of myocardial or other tissues; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders; thermal injury; hemodialysis;leukapheresis; ulcerative colitis; Crohn's disease; necrotizingenterocolitis; granulocyte transfusion associated syndromes; andcytokine-induced toxicity.

As discussed above, the binding of ICAM-2 molecules to the members ofCD-18 family of molecules is of central importance in cellular adhesion.Through the process of adhesion, lymphocytes are capable of continuallymonitoring an animal for the presence of foreign antigens. Althoughthese processes are normally desirable, they are also the cause of organtransplant rejection, tissue graft rejection and many autoimmunediseases. Hence, any means capable of attenuating or inhibiting cellularadhesion would be highly desirable in recipients of organ transplants(especially kidney transplants), tissue grafts, or for autoimmunepatients.

Monoclonal antibodies to members of the CD-18 family inhibit manyadhesion dependent functions of leukocytes including binding toendothelium (Haskard, D. et al., J. Immunol. 137:2901-2906 (1986)),homotypic adhesions (Rothlein, R. et al., J. Exp. Med. 163:1132-1149(1986)), antigen and mitogen induced proliferation of lymphocytes(Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)),antibody formation (Fischer, A. et al., J. Immunol. 136:3198-3203(1986)), and effector functions of all leukocytes such as lytic activityof cytotoxic T cells (Krensky, A. M. et al., J. Immunol. 132:2180-2182(1984)), macrophages (Strassman, G. et al., J. Immunol. 136:4328-4333(1986)), and all cells involved in antibody-dependent cellularcytotoxicity reactions (Kohl, S. et al., J. Immunol. 133:2972-2978(1984)). In all of the above functions, the antibodies inhibit theability of the leukocyte to adhere to the appropriate cellular substratewhich in turn inhibits the final outcome. Such functions, to the extentthat they involve ICAM-2/LFA-1 interactions, can be suppressed withanti-ICAM-2 antibody.

Thus, monoclonal antibodies capable of binding to ICAM-2 can be employedas anti-inflammatory agents in a mammalian subject. Significantly, suchagents differ from general anti-inflammatory agents in that they arecapable of selectively inhibiting adhesion, and do not offer other sideeffects such as nephrotoxicity which are found with conventional agents.

Since ICAM-2, particularly in soluble form is capable of acting in thesame manner as an antibody to members of the CD-18 family, it may beused to suppress inflammation. Moreover, the functional derivatives andantagonists of ICAM-2 may also be employed to suppress inflammation.

1. Suppressors of Delayed Type Hypersensitivity Reactions

ICAM-2 molecules mediate, in part, adhesion events necessary to mountinflammatory reactions such as delayed type hypersensitivity reactions.Thus, antibodies (especially monoclonal antibodies) capable of bindingto ICAM-2 molecules have therapeutic potential in the attenuation orelimination of such reactions.

Alternatively, since ICAM-2 is an antagonist of the ICAM-1/LFA-1interaction, ICAM-2 (particularly in solublilized form), or itsfunctional derivatives can be used to suppress delayed typehypersensitivity reactions.

These potential therapeutic uses may be exploited in either of twomanners. First, a composition containing a monoclonal antibody againstICAM-2. may be administered to a patient experiencing delayed typehypersensitivity reactions. For example, such compositions might beprovided to a individual who had been in contact with antigens such aspoison ivy, poison oak, etc. In the second embodiment, the monoclonalantibody capable of binding to ICAM-2 is administered to a patient inconjunction with an antigen in order to prevent a subsequentinflammatory reaction. Thus, the additional administration of an antigenwith an ICAM-2-binding monoclonal antibody may temporarily tolerize anindividual to subsequent presentation of that antigen.

2. Therapy for Chronic Inflammatory Disease

Since LAD patients that lack LFA-1 do not mount an inflammatoryresponse, it is believed that antagonism of LFA-1's natural ligand,ICAM-2, will also inhibit an inflammatory response. The ability ofantibodies against ICAM-2 to inhibit inflammation provides the basis fortheir therapeutic use in the treatment of chronic inflammatory diseasesand autoimmune diseases such as lupus erythematosus, autoimmunethyroiditis, experimental allergic encephalomyelitis (EAE), multiplesclerosis, some forms of diabetes, Reynaud's syndrome, rheumatoidarthritis, etc. Such antibodies may also be employed as a therapy in thetreatment of psoriasis. In general, the monoclonal antibodies capable ofbinding to ICAM-2 may be employed in the treatment of those diseasescurrently treatable through steroid therapy.

In accordance with the present invention, such inflammatory and immunerejection responses may be suppressed (i.e. either prevented orattenuated) by providing to a subject in need of such treatment anamount of an anti-inflammatory agent sufficient to suppress saidinflammation. Suitable anti-inflammatory agents include: an antibodycapable of binding to ICAM-2; a fragment of an antibody, which fragmentis capable of binding to ICAM-2; ICAM-2; a functional derivative ofICAM-2; a non-immunoglobulin antagonist of ICAM-2 other than ICAM-1 or anon-immunoglobulin antagonist of ICAM-2 other than LFA-1. Especiallypreferred are anti-inflammatory agents composed of a soluble functionalderivative of ICAM-2. Such anti-inflammatory treatment can also includethe additional administration of an agent selected from the groupconsisting of: an antibody capable of binding to LFA-1; a functionalderivative of an antibody, said functional derivative being capable ofbinding to LFA-1; and a non-immunoglobulin antagonist of LFA-1.

The invention further includes the above-described methods forsuppressing an inflammatory response of the specific defense system inwhich an immunosuppressive agent is additionally provided to thesubject. Such an agent is preferably provided at a dose lower (i.e. a“sub-optimal” dose) than that at which it would normally be required.The use of a sub-optimal dose is possible because of the synergisticeffect of the agents of the present invention. Examples of suitableimmunosuppressive agents include dexamethesone, azathioprine, ICAM-1,cyclosporin A, etc.

3. Therapy for Non-Specific Inflammation

The present invention derives in part from the discovery thatgranulocyte-endothelial cell adherence results from the interaction ofglycoproteins of the CD-18 family with the endothelium. Since cellularadhesion is required in order that leukocytes may migrate to sites ofinflammation and/or carry out various effector functions contributing toinflammation, agents which inhibit cellular-adhesion will attenuate orprevent such inflammation. Such inflammatory reactions are due toreactions of the “non-specific defense system” which are mediated byleukocytes incapable of immunological memory. Such cells includegranulocytes and macrophages. As used herein, inflammation is said toresult from a response of the non-specific defense system, if theinflammation is caused by, mediated by, or associated with a reaction ofthe non-specific defense system. Examples of inflammation which result,at least in part, from a reaction of the non-specific defense systeminclude inflammation associated with conditions such as: adultrespiratory distress syndrome (ARDS) or multiple organ injury syndromessecondary to septicemia or trauma; reperfusion injury of myocardial orother tissues; acute glomerulonephritis; reactive arthritis; dermatoseswith acute inflammatory components; acute purulent meningitis or othercentral nervous system inflammatory disorders; thermal injury;hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease;necrotizing enterocolitis; granulocyte transfusion associated syndromes;and cytokine-induced toxicity.

The anti-inflammatory agents of the present invention are compoundscapable of specifically antagonizing the interaction of the CD-18complex on granulocytes with endothelial cells. Such antagonistscomprise: ICAM-2; a functional derivative of ICAM-2; and anon-immunoglobulin antagonist of ICAM-2 other than ICAM-1, or a memberof the CD-18 family of molecules.

B. Suppressors of Organ and Tissue Rejection

Since ICAM-2, particularly in soluble form is capable of acting in thesame manner as an antibody to members of the CD-18 family, it may beused to suppress organ or tissue rejection caused by any of the cellularadhesion-dependent functions. Moreover, anti-ICAM-2 antibody and thefunctional derivatives and antagonists of ICAM-2 may also be employed tosuppress such rejection.

ICAM-2 and antibodies capable of binding to ICAM-2 can be used toprevent organ or tissue rejection, or modify autoimmune responseswithout the fear of such side effects, in the mammalian subject.

Importantly, the use of monoclonal antibodies capable of recognizingICAM-2 may permit one to perform organ transplants even betweenindividuals having HLA mismatch.

C. Adjunct to the Introduction of Antigenic Material Administered forTherapeutic or Diagnostic Purposes

Immune responses to therapeutic or diagnostic agents such as, forexample, bovine insulin, interferon, tissue-type plasminogen activatoror murine monoclonal antibodies substantially impair the therapeutic ordiagnostic value of such agents, and can, in fact, causes diseases suchas serum sickness. Such a situation can be remedied through the use ofthe antibodies of the present invention. In this embodiment, suchantibodies would be administered in combination with the therapeutic ordiagnostic agent. The addition of the antibodies prevents the recipientfrom recognizing the agent, and therefore prevents the recipient frominitiating an immune response against it. The absence of such an immuneresponse results in the ability of the patient to receive additionaladministrations of the therapeutic or diagnostic agent.

ICAM-2 (particularly in solubilized form) or its functional derivativesmay be employed interchangeably with ICAM-1, or with antibodies capableof binding to LFA-1 in the treatment of disease. Thus, in solubilizedform, such molecules may be employed to inhibit organ or graftrejection. ICAM-2, or its functional derivatives may be used in the samemanner as anti-ICAM-2 antibodies to decrease the immunogenicity oftherapeutic or diagnostic agents.

D. Suppressors of Tumor Metastasis

The agents of the present invention may also be employed to suppress themetastasis of a hematopoietic tumor cell, which requires a functionalmember of the CD-18 family for migration. In accordance with thisembodiment of the present invention, a patient in need of such treatmentis provided with an amount of an agent (such as an antibody capable ofbinding to ICAM-2; a toxin-derivatized antibody capable of binding toICAM-2; a fragment of an antibody, the fragment being capable of bindingto ICAM-2; a toxin-derivatized fragment of an antibody, the fragmentbeing capable of binding to ICAM-2; ICAM-2; a functional derivative ofICAM-2; and a non-immunoglobulin antagonist of ICAM-2 other than ICAM-1)sufficient to suppress said metastasis.

The invention also provides a method of suppressing the growth of anICAM-2-expressing tumor cell which comprises providing to a patient inneed of such treatment an amount of an agent sufficient to suppress saidgrowth. Suitable agents include an antibody capable of binding toICAM-2; a toxin-derivatized antibody capable of binding to ICAM-2; afragment of an antibody, the fragment being capable of binding toICAM-2; a toxin-derivatized fragment of an antibody, the fragment beingcapable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2;a non-immunoglobulin antagonist of ICAM-2 other than ICAM-1; atoxin-derivatized member of the CD-18 family of molecules; and atoxin-derivatized functional derivative of a member of the CD-18 familyof molecules.

The invention also provides a method of suppressing the growth of anLFA-1-expressing tumor cell which comprises providing to a patient inneed of such treatment an amount of a toxin sufficient to suppress saidgrowth. Suitable toxins include a toxin-derivatized ICAM-2, or atoxin-derivatized functional derivative of ICAM-2.

E. Suppressors of Viral Infection

ICAM-1 has recently been shown to be subverted as a receptor by themajor group of rhinoviruses (Greve, J. M. et al., Cell 56:839-847(1989); Staunton, D. E. et al., Cell 56:849-853 (1989); Tomassini, J. E.et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4907-4911 (1989), whichreferences are incorporated herein by reference). Rhinoviruses, membersof the small, RNA-containing, protein-encapsidated picornavirus family,cause 40-50% of common colds (Rueckert, R. R., In: Fields Virology,Fields, B. N. et al. (eds.), Raven Press, NY, (1985) pp 705-738;Sperber, S. J. et al. Antimicr. Agents Chemo. 32: 409-419 (1988), whichreferences are incorporated herein by reference). Over 100immunologically non-crossreactive rhinoviruses have been defined, ofwhich 90% bind to ICAM-1.

Besides ICAM-1, the cell adhesion molecule CD4 and the complementreceptor CR2 have recently been found to be subverted as virus receptorsby HIV and EBV viruses, respectively (Maddon, P. J., Cell 47:333-348(1986); Fingeroth, J. D., et al., Proc. Natl. Acad. Sci. USA81:4510-4514 (1984), which references are incorporated herein byreference). Further, a molecule with an Ig domain structure similar toICAM-1 and which may function in cellular adhesion is a polio virusreceptor (Mendelsohn, C. L., et al., Cell 56:855-865 (1989)).

ICAM-2 and its functional derivatives may act as receptors for viral(particularly by rhinoviruses, and particularly rhinoviruses of theminor serotype) attachment or infection. Thus, antibody to ICAM-2 (orfragments thereof), ICAM-2, or functional derivatives of ICAM-2, may beemployed to block such attachment or infection, and to thereby suppressviral infection.

F. Diagnostic and Prognostic Applications

Monoclonal antibodies capable of binding to ICAM-2 may be employed as ameans of imaging or visualizing the sites of ICAM-2 expression andinflammation in a patient. In such a use, the monoclonal antibodies aredetectably labeled, through the use of radioisotopes, affinity labels(such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms,labeled anti-ICAM-2 antibody, etc. Procedures for accomplishing suchlabeling are well known to the art. Clinical application of antibodiesin diagnostic imaging are reviewed by Grossman, H. B., Urol. Clin. NorthAmer. 13:465-474 (1986)), Unger, E. C. et al., Invest. Radiol.20:693-700 (1985)), and Khaw, B. A. et al., Science 209:295-297 (1980)).

The presence of ICAM-2 expression may also be detected through the useof binding ligands, such as mRNA, cDNA, or DNA which bind to ICAM-2 genesequences, or to ICAM-2 mRNA sequences, of cells which express ICAM-2.Techniques for performing such hybridization assays are described byManiatis, T. et al., In: Molecular Cloning, a Laboratory Manual,Coldspring Harbor, N.Y. (1982), and by Haymes, B. D. et al., In: NucleicAcid Hybrization, a Practical Approach, IRL Press, Washington, D.C.(1985), which references are herein incorporated by reference.

The detection of foci of such detectably labeled antibodies isindicative of a site of ICAM-2 expression or tumor development. In oneembodiment, this examination for expression is done by removing samplesof tissue or blood and incubating such samples in the presence ofantibodies which are or which can be detectably labeled. In a preferredembodiment, this technique is done in a non-invasive manner through theuse of magnetic imaging, fluorography, etc. Such a diagnostic test maybe employed in monitoring organ transplant recipients for early signs ofpotential tissue rejection. Such assays may also be conducted in effortsto determine an individual's predilection to rheumatoid arthritis orother chronic inflammatory diseases.

For example, by radioactively labeling the antibodies or antibodyfragments, it is possible to detect the antigen through the use ofradioimmune assays. A good description of a radioimmune assay (RIA) maybe found in Laboratory Techniques and Biochemistry in Molecular Biology,by Work, T. S., et al., North Holland Publishing Company, NY (1978),with particular reference to the chapter entitled “An Introduction toRadioimmune Assay and Related Techniques” by Chard, T., incorporated byreference herein. Alternatively, flouresecent, enzyme, or other suitablelabels can be employed.

Examples of types of labels which can be used in the present inventioninclude, but are not limited to, enzyme labels, radioisotopic labels,non-radioactive isotopic labels, fluorescent labels, toxin labels, andchemiluminescent labels.

Examples of suitable enzyme labels include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,etc.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. Examples of suitable non-radioactiveisotopic labels include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, ⁵⁶Fe, etc.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an o-phthaldehyde label, afluorescamine label, etc.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, an aequorin label, etc.

VI. Administration of the Compositions of the Present Invention

The therapeutic effects of ICAM-2 may be obtained by providing to apatient the entire ICAM-2 molecule, or any therapeutically activepeptide fragments thereof. Of special interest are therapeuticallyactive peptide fragments of ICAM-2 which are soluble.

ICAM-2 and its functional derivatives may be obtained eithersynthetically, through the use of recombinant DNA technology, or byproteolysis, or by a combination of such methods. The therapeuticadvantages of ICAM-2 may be augmented through the use of functionalderivatives of ICAM-2 possessing additional amino acid residues added toenhance coupling to carrier or to enhance the activity of the ICAM-2.The scope of the present invention is further intended to includefunctional derivatives of ICAM-2 which lack certain amino acid residues,or which contain altered amino acid residues, so long as suchderivatives posess or affect a biological or pharmacological activity ofICAM-2.

Both the antibodies of the present invention and the ICAM-2 moleculedisclosed herein are said to be “substantially free of naturalcontaminants” if preparations which contain them are substantially freeof materials with which these products are normally and naturally found.

The present invention extends to antibodies, and biologically activefragments thereof, (whether polyclonal or monoclonal) which are capableof binding to ICAM-2. Such antibodies may be produced either by ananimal, or by tissue culture, or recombinant DNA means.

In providing a patient with antibodies, or fragments thereof, capable ofbinding to ICAM-2, or when providing ICAM-2 (or a fragment, variant, orderivative thereof) to a recipient patient, the dosage of administeredagent will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition, previous medicalhistory, etc. In general, it is desirable to provide the recipient witha dosage of antibody which is in the range of from about 1 pg/kg to 10mg/kg (body weight of patient), although a lower or higher dosage may beadministered. When providing ICAM-2 molecules or their functionalderivatives to a patient, it is preferable to administer such moleculesin a dosage which also ranges from about 1 pg/kg to 10 mg/kg (bodyweight of patient) although a lower or higher dosage may also beadministered. As discussed below, the therapeutically effective dose canbe lowered if the anti-ICAM-2 antibody is additionally administered withan anti-LFA-1 antibody. As used herein, one compound is said to beadditionally administered with a second compound when the administrationof the two compounds is in such proximity of time that both compoundscan be detected at the same time in the patient's serum.

Both the antibody capable of binding to ICAM-2 and ICAM-2 itself may beadministered to patients intravenously, intramuscularly, subcutaneously,enterally, or parenterally. When administering antibody or ICAM-2 byinjection, the administration may be by continuous infusion, or bysingle or multiple boluses.

The agents of the present invention are intended to be provided torecipient subjects in an amount sufficient to suppress inflammation. Anamount is said to be sufficient to “suppress” inflammation if thedosage, route of administration, etc. of the agent are sufficient toattenuate or prevent inflammation.

Anti-ICAM-2 antibody, or a fragment thereof, may be administered eitheralone or in combination with one or more additional immunosuppressiveagents (especially to a recipient of an organ or tissue transplant). Theadministration of such compound(s) may be for either a “prophylactic” or“therapeutic” purpose. When provided prophylactically, theimmunosuppressive compound(s) are provided in advance of anyinflammatory response or symptom (for example, prior to, at, or shortlyafter) the time of an organ or tissue transplant but in advance of anysymptoms of organ rejection). The prophylactic administration of thecompound(s) serves to prevent or attenuate any subsequent inflammatoryresponse (such as, for example, rejection of a transplanted organ ortissue, etc.). When provided therapeutically, the immunosuppressivecompound(s) is provided at (or shortly after) the onset of a symptom ofactual inflammation (such as, for example, organ or tissue rejection).The therapeutic administration of the compound(s) serves to attenuateany actual inflammation (such as, for example, the rejection of atransplanted organ or tissue).

The anti-inflammatory agents of the present invention may, thus, beprovided either prior to the onset of inflammation (so as to suppress ananticipated inflammation) or after the initiation of inflammation.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

The antibody and ICAM-2 molecules of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human-serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack,Easton Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of anti-ICAM-2 antibody or ICAM-2molecule, or their functional derivatives, together with a suitableamount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb anti-ICAM-2 antibody or ICAM-2,or their functional derivatives. The controlled delivery may beexercised by selecting appropriate macromolecules (for examplepolyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine, sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate anti-ICAM-2 antibody or ICAM-2 molecules,or their functional derivatives, into particles of a polymeric materialsuch as polyesters, polyamino acids, hydrogels, poly(lactic acid) orethylene vinylacetate copolymers. Alternatively, instead ofincorporating these agents into polymeric particles, it is possible toentrap these materials in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxy-methylcellulose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

The invention further includes a phamaceutical composition comprising:(a) an anti-inflammatory agent (such as an antibody capable of bindingto ICAM-2; a fragment of an antibody, said fragment being capable ofbinding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; and anon-immunoglobulin antagonist of ICAM-2 other than ICAM-1, and (b) atleast one immunosuppressive agent. Examples of suitableimmunosuppressive agents include: dexamethesone, azathioprine andcyclosporin A.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Cloning of ICAN-2 cDNA

In order to clone cDNA capable of encoding ICAM-2, a modification of theprocedure of Aruffo and Seed (Seed, B. et al., Proc. Natl. Acad. Sci.USA 84:3365-3369 (1987)) for selecting cDNAs by expression in COS cellswas employed to pan for ligand-bearing COS cells on functionally-active,purified LFA-1 bound to plastic Petri dishes.

In detail, LFA-1 was purified from SKW-3 lysate by immunoaffinitychromatography on TS2/4 LFA-1 MAb Sepharose and eluted at pH 11.5 in thepresence of 2 mM MgCl₂ and 1% octylglucoside. LFA-1 (10 μg/200 μl /6 cmplate) was bound to bacteriological Petri dishes by dilutingoctylglucoside to 0.1in PBS with 2 mM MgCl₂ and overnight incubation at4° C. Plates were blocked with 1% BSA and stored in PBS/2 mM MgCl₂/0.2%BSA/0.025% azide/50 μg/ml gentamycin.

Synthesis of a CDNA library from LPS-stimulated umbilical veinendothelial cells by the method of Gubler and Hoffman was performed asdescribed by Staunton et al. (Staunton, D. E. et al., Cell 52:925-933(1988)). Following second strand synthesis the cDNA was ligated to BstX1 adaptors (Seed, B. et al., Proc. Natl. Acad. Sci. USA 84:3365-3369(1987)) and cDNA's>600 bp were selected by low melting point (LMP)agarose gel electrophoresis. The cDNA was then ligated to CDM8 (Seed,B., Nature 329:840-842 (1987)), introduced into E. coli host MC1061/P3and plated to obtain 5×10⁵ colonies. The colonies were suspended in LBmedium, pooled and plasmid prepared by standard alkali lysis method(Maniatis, T. et al., in Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (1982)). Ten 10 cm plates of COS cells at 50%confluency were transfected with 10 μg/plate of the plasmid cDNA libraryusing DEAE-dextran (Kingston, R. E., in Curent Protocols in MolecularBiology, 9.0.1-9.9.6, Greene Publishing Associates (1987)). ICAM-2 istrypsin-resistant on endothelial and SKW-3 cells. COS cells three dayspost transfection were suspended by treatment with 0.025% trypsin/1 mMEDTA/HBSS (Gibco) and panned (Seed, B. et al., Proc. Natl. Acad. Sci.USA 84:3365-3369 (1987)) on LFA-1 coated plates as described below for⁵¹Cr-labelled COS cells. Adherent cells were released by addition ofEDTA to 10 mM.

Plasmid was recovered from the adherent population of COS cells in Hirtsupernatants (Hirt, B. J., J. Mol. Biol. 26:365-369 (1967)). The E. colistrain MC1061/P3 was then transformed with the plasmid, colonies onplates were suspended in LB medium, pooled and plasmid prepared byalkali-lysis method. Selection of LFA-1-adherent transfected COS cellsand plasmid recovery was repeated for two more cycles. Pooled coloniesobtained after the third cycle were grown to saturation in 100 ml of LBmedium with 18 μg/ml tetracycline and 20 μg/ml ampicillin. Plasmid wasprepared and fractionated by 1% LMP-agarose gel electrophoresis andMC1061/p3 was transformed separately with plasmid from nine differentsize fractions. Individual plasmids from the fraction with greatestactivity in promoting COS cell adhesion to LFA-1 were examined forinsert size by digestion with Xbal and tested in the COS cell adherenceassay. This yielded one plasmid with an-ICAM-2 cDNA insert of 1.1 kb,pCDIC2.27.

For adhesion assays, the ICAM-2 plasmid pCDIC2.27 or an ICAM-1 constructcontaining the 1.8 kb SalI, KpnI fragment (Staunton, D. E. et al., Cell52:925-933 (1988)) in CDM8 (2 μg/10 cm plate) were introduced into COScells using DEAE-Dextran. COS cells were suspended with 0.025% trypsin/1mM EDTA/HBSS three days post transfection and labelled with ⁵¹Cr.Approximately 2×10⁵ ⁵¹Cr labelled COS cells in 2 ml PBS/5% FCS/2 mMMgCl₂/0.025% azide (buffer) with 5 μg/ml of the MAb indicated wereincubated in LFA-1-coated 6 cm plates at 25° C. for 1 hour. Non-adherentcells were removed by gentle rocking and three washes with buffer.Adherent cells were eluted by the addition of EDTA to 10 mM andγ-counted.

The feasibility of this procedure was demonstrated using COS cellstransfected with the previously cloned ICAM-1 CDNA (FIG. 1A). ICAM-1 wasexpressed on 25% of the transfected COS cells. After panning,nonadherent cells were depleted of ICAM-1⁺ cells, whereas adherent cellsreleased from LFA-1-coated plastic by EDTA were almost completelyICAM-1⁺. Adherence of ICAM-1⁺ cells to LFA-1-coated plastic wasinhibited with RR1/1 ICAM-1 MAb. LFA-1-coated on Petri dishes was stableto >5 cycles of COS cell adherence and elution with EDTA; plates werestored with Mg²⁺ at 4° C. in between use.

To clone ICAM-2, a cDNA library in the plasmid vector CDM8 was preparedfrom endothelial cells, which demonstrate both the ICAM-1-dependent andICAM-1-independent components of LFA-1-dependent adhesion (Dustin, M. L.et al., J. Cell. Biol. 107:321-331 (1988)). Transfected COS cells wereincubated in LFA-1-coated petri dishes with ICAM-1 MAb present toprevent isolation of ICAM-1 cDNA's. Adherent cells were eluted with EDTAand plasmids were isolated and amplified in E. coli. Following threecycles of transfection, adherence, and plasmid isolation; and one sizefractionation, 30 plasmids were analyzed by restriction endonucleasedigestion. Of three with inserts>1.0 kb, one plasmid introduced into COScells by transfection yielded adherence to LFA-1.

The isolated plasmid conferred adherence to LFA-1 on a high percentageof the transfected cells, similar to the percentage seen with ICAM-1transfection (FIG. 1B). Adherence was blocked by LFA-1 mAb, but incontrast to ICAM-1 transfectants, not by ICAM-1 mAb (FIG. 1B).Futhermore, cells transfected with this plasmid did not react with apanel of four ICAM-1 mAb. Thus, all functional criteria for a cDNAencoding a second LFA-1 ligand were fulfilled, and the ligand wasdesignated “ICAM-2.”

EXAMPLE 2 Characterization of ICAM-2 cDNA Sequence

The ICAM-2 cDNA sequence of 1052 bp (FIGS. 2-1, 2-2, 2-3, and 2-4)contains a 62 bp 5′ and a 167 bp 3′ untranslated region. An AATACApolyadenylation signal at position 1019, which in contrast to AATAAA,occurs in approximately 2% of vertebrate mRNAs (Wickens, M. et al.,Science 226:1045-1051 (1984)), is followed at 1058 bp by a poly(A) tail.The longest open reading frame begins with the first ATG at position 63and ends with a TAG termination codon at position 885. Hydrophobicityanalysis (Kyte, J. et al., J. Mol. Biol. 157:105-132 (1982)) and usageof amino acids around cleavage sites (von Heijne, G., Nucleic AcidsResearch 14:4683-4690 (1986)) predict a 21 residue signal peptide (FIG.2-1).

The predicted mature sequence contains from amino acid 1 to 201 aputative extracellular domain followed by a 26 residue hydrophobicputative transmembrane domain and a 26 residue cytoplasmic domain. Fourturns of the putatively α-helical transmembrane segment are amphipathic,with threonine and serine residues falling on one side, suggesting thepossibility of self-association or association with other membraneproteins in the plane of the membrane. The cytoplasmic domain isunusually basic, and in contrast to most cytoplasmic domains which arehydrophilic, is of average hydrophobicity. The predicted mass of themature polypeptide is 28,176 daltons which, if the six predictedN-linked glycosylation sites are used, would result in a ICAM-2glycoprotein of approximately 46 Kd.

EXAMPLE 3 DNA and RNA Hybridization Analyses

The isolated ICAM-2 cDNA clones were analyzed using both Northern andSouthern hybridization. Northern blots used 6 μg of poly(A)⁺ RNA whichwas denatured and electrophoresed through a 1% agarose-formal-dehyde gel(Maniatis, T. et al., in Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (1982)) and electrotransferred to a nylonmembrane (Zeta Probe, BioRad). Completion of transfer was confirmed byUV trans-illumination of the gel and fluorescent photography of theblot.

The genomic DNAs were digested with five times the manufacturer'srecommended quantity of EcoRI and HindIII endonucleases (New EnglandBiolabs). Following electrophoresis through a 0.8% agarose gel, the DNAswere transferred to ZETA PROBE. RNA and DNA blots were prehybridized andhybridized following standard procedures (Maniatis, T. et al., inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory(1982)) using ICAM-2 or ICAM-1 cDNAs labeled with α[³²P]d XTP's byrandom priming (Boehringer Mannheim).

The 1.1 kb ICAM-2 CDNA hybridizes to a 1.4 kb poly(A)⁺ mRNA and weaklyto a 3 kb mRNA (FIG. 3A), distinct from the 3.3 kb and 2.4 kb ICAM-1mRNA (FIG. 3B). mRNA was examined in cells which have been characterizedfunctionally for ICAM-1-dependent and second ligand-dependent binding toLFA-1. ICAM-1 mRNA is strongly induced in endothelial cells by LPS (FIG.3B, lanes 2 and 3). In contrast, ICAM-2 mRNA is strongly expressedbasally in endothelial cells and is not induced further by LPS (FIG. 3A,lanes 2 and 3). This correlates with strong basal and non-inducibleexpression of the LFA-1-dependent, ICAM-1-independent pathway inendothelial cells and inducibility of the ICAM-1-dependent pathway(Dustin, M. L. et al., J. Cell. Biol. 107:321-331 (1988)).

ICAM-2 mRNA is present in a wide variety of cell types including Ramosand BBN B lymphoblastoid, U937 monocytic, and SKW3 lymphoblastoid celllines (FIG. 3A, lanes 1,4,6, and 8), as shown by moderate or longautoradiogram exposure. Of these, SKW3, U937, and BBN have been shown toexhibit LFA-1-dependent, ICAM-1-independent adhesion to LFA-1⁺ cells(Rothlein, R. et al., J. Immunol. 137:1270-1274 (1986); Makgoba, M. W.et al., Eur. J. Immunol. 18:637-640 (1988)), and to LFA-1-coatedplastic. The HeLa epithelial cell line, which exhibits only theICAM-1-dependent component of LFA-1-dependent adhesion (Makgoba, M. W.et al., Eur. J. Immunol. 18:637-640 (1988)), shows no ICAM-2 mRNA (FIG.3A, lane 5), even after prolonged autoradiogram exposure. The celldistribution of ICAM-2 is thus consistent with the ICAM-1-independentcomponent of LFA-1-dependent adhesion.

Southern blots of genomic DNA (FIG. 3D) hybridized with the ICAM-2 cDNAshowed a single predominant EcoRI fragment of 8.2 kb and HindIIIfragment of 14 kb, suggesting a single gene with most of the codinginformation present in 8 kb.

EXAMPLE 4 Comparison of the Amino Acid Sequences of ICAM-1 and ICAM-2

Because of their functional similarity as LFA-1 ligands, the amino acidsequences of ICAM-2 and ICAM-1 were compared. ICAM-1 is a member of theIg superfamily and its extracellular domain consists entirely of fiveC-like domains. The 201 amino acid extracellular domain of ICAM-2consists of 2 Ig C-like domains, with putative intradomaindisulfide-bonded cysteines spaced 43 and 56 residues apart and apredicted β strand structure (FIGS. 4-1 and 4-2). Remarkably, the twoIg-like like domains of ICAM-2 are 34% identical in amino acid sequenceto the two most N-terminal Ig-like domains of ICAM-1 (FIGS. 4-1 and4-2), with an ALIGN score 15 s.d. above the mean, and 27% identical toICAM-1 domains 3 and 4, with an ALIGN score 3 s.d. above the mean.

Search of the NBRF and SWISS-PROT protein databases yielded only partialdomain homologies with other members of the Ig superfamily, primarilywith HLA Class II antigens. ICAM-2 shows somewhat fewer conservedresidues characteristic of Ig domains than ICAM-1. ICAM-2 is 17% and 19%identical to the two N-terminal domains of the adhesion molecules NCAM(Cunningham, B. A. et al., Science 236:799-806 (1987)) and MAG (Salzer,J. L. et al., J. Cell Biol. 104:957-965 (1987)), respectively, whileICAM-1 is 19% and 20% identical, respectively.

Lymphocyte function associated antigen-1 (LFA-1) and intercellularadhesion molecule-1 (ICAM-1) were identified by selecting MAb whichblocked T lymphocyte-mediated killing, and homotypic adhesion,respectively (Rothlein, R. et al., J. Immunol. 137:1270-1274 (1986);Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)). Incontrast, ICAM-2 has been defined using a functional cDNA selectionprocedure which requires no previous identification of the protein bybiochemical or immunological techniques.

Isolation of a cDNA for ICAM-2 confirms the postulated existence of analternative LFA-1 ligand. The distribution of mRNA for ICAM-2 on alimited number of cells which have been characterized forICAM-1-dependent and ICAM-1-independent adhesion to LFA-1 suggests thatICAM-2 could account for all of the observed ICAM-1-independentLFA-1-dependent adhesion.

ICAM-2 and the two N-terminal domains of ICAM-1 are much more like oneanother than like other members of the Ig superfamily, demonstrating asubfamily of Ig-like molecules which bind to LFA-1. Significantly, theLFA-1-binding region of ICAM-1 has been mapped to domains 1 and 2 bydomain deletion and systematic amino acid substitution. Thus, there isboth structural and functional homology. ICAM-2 is the second example ofan Ig-family member which binds to an integrin. Although there is littleprecedence among cell adhesion receptors, among the integrins a numberof receptors for extracellular matrix components have been shown torecognize multiple ligands (Hynes, R. O., Cell 48:549-554 (1987);Ruoslahti, E. et al., Science 238:491-497 (1987)).

Neither ICAM-1 or ICAM-2 contains an RGD sequence, and thus the mode ofrecognition by LFA-1 may differ from integrins which bind extracellularmatrix components (Hynes, R. O., Cell 48:549-554 (1987); Ruoslahti, E.et al., Science 238:491-497 (1987)). The cellular ligands recognized byMac-1 and p150,95, leukocyte integrins closely related to LFA-1, maybelong to the same Ig subfamily. ICAM-1 has recently been demonstratedto be a receptor for the major group of rhinoviruses which cause 50% ofcommon colds. ICAM-2 may also function as a receptor for rhinoviruses orother piconaviruses. Thus, it may be used in a therapy to suppress (i.e.prevent or attenuate) infection from such viruses.

A family of ligands for LFA-1 emphasizes the importance of thisrecognition pathway and may be a mechanism for imparting finespecificity and functional diversity. A number of differences betweenICAM-1 and ICAM-2 are of potential importance. ICAM-1 is inducible onmost cells while ICAM-2 expression is not affected by cytokines on thecells thus far tested. The three additional domains on ICAM-1 areexpected to project its LFA-1 binding site further from the cell surfacethan that of ICAM-2, suggesting that closer cell-cell contact would berequired for LFA-1:ICAM-2 than LFA-1:ICAM-1 interaction. ICAM-2transfected COS cells are more readily detached than ICAM-1 transfectedCOS cells from LFA-1 coated plastic as the washing shear force isincreased. This may be due to the smaller size of ICAM-2 which may makeit less accessible to LFA-1 on the artificial substrate, or todifferences in sequence which impart differences in affinity.

The distinct cytoplasmic domains of ICAM-1 and ICAM-2 may impartdifferent signals or may cause differing localization on the cellsurface; likewise, signalling or interaction with the cytoskeleton byLFA-1 may differ depending on whether ICAM-1 or ICAM-2 is bound.

ICAM-1 and a second LFA-1 counter-receptor, ICAM-2, thus constitute asubfamily of the immunoglobulin (1g) superfamily (Staunton, D. E., etal., Cell 52:925-933 (1988), which reference is incorporated herein byreference). ICAM-1 possesses five Ig-like C domains whereas ICAM-2possesses two, which are most homologous to the amino terminal domainsof ICAM-1. ICAM-1 and ICAM-2, expressed on a variety of cell types,support various leukocyte adhesion dependent functions includinginduction and effector functions in the immune response. ICAM-1expression is highly inducible by cytokines and thus the LFA-1/ICAM-1adhesion system is able to guide leukocyte migration and localizationduring inflammation (Rothlein, R. J. Immunol. 137:1270-1274 (1986);Marlin, S. D. et al., Cell 51:813-819 (1987); Kishimoto, T. K. et al.,Adv. Immunol. 46:149-182 (1989); Dustin, M. L. et al., Immunol. Today9:213-215 (1988), all of which references are incorporated herein byreference).

ICAM-1 residues which have been defined above as being important toLFA-1 binding are conserved in other ICAMs (Staunton, D. E., et al.Nature 339:61-64 (1989), which reference is incorporated herein byreference). Human ICAM-1 is 50% identical to murine ICAM-1 and 35%identical to human ICAM-2 (Staunton, D. E., et al. Nature 339:61-64(1989). The residues that are most critical to LFA-1 binding, E34 andQ73, are conserved both in mouse ICAM-1 and in human ICAM-2. This isconsistent with the ability of both mouse ICAM-1 and human ICAM-2(Staunton, D. E., et al. Nature 339:61-64 (1989)) to bind to humanLFA-1. One D2 N-linked glycosylation site at N156, which influencesLFA-1 binding, is also conserved in ICAM-2. Several residues that areimportant to rhinovirus-14 binding, Q58, G46, D71, K77 and R166, are notconserved in mouse ICAM-1 or human ICAM-2 (Staunton, D. E. et al., Cell56:849-853 (1989), which reference is incorporated herein by reference)which is consistent with the apparent inability of mouse cells (Colonno,R. J. et al., J. Virol. 57:7-12 (1986)) and ICAM-2 to bindrhinovirus-14.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

What is claimed is:
 1. A method for treating inflammation in a mammaliansubject which comprises providing to a subject in need of such treatmentan amount of anti-inflammatory agent sufficient to suppress saidinflammation; wherein said inflammation involves cellular adhesionmediated by ICAM-2, and said anti-inflammatory agent is capable ofsuppressing said cellular adhesion and is selected from the groupconsisting of: an antibody capable of binding to ICAM-2; a fragment ofan antibody, said fragment being capable of binding to ICAM-2; andICAM-2.
 2. The method of claim 1, wherein said inflammation is inresponse to a condition selected from the group consisting of: delayedtype hypersensitivity reaction, a symptom of psoriasis, an autoimmunedisease, organ transplant or tissue graft rejection.
 3. The method ofclaim 2, wherein said autoimmune disease is selected from the groupconsisting of: Reynaud's syndrome, autoimmune thyroiditis, EAE, multiplesclerosis, rheumatoid arthritis and lupus erythematosus.
 4. The methodof claim 1, wherein said inflammation is in response to a conditionselected from the group consisting of: adult respiratory distresssyndrome (ARDS), multiple organ injury syndromes secondary to septicemiaor trauma, reperfusion injury of myocardial or other tissues, acuteglomerulonephritis, reactive arthritis, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, thermal injury, hemodialysis,leukapheresis, ulcerative colitis, Crohn's disease, necrotizingenterocolitis, granulocyte transfusion associated syndromes, andcytokine-induced toxicity.